Oligoribonucleotides and methods of use thereof for treatment of alopecia, acute renal failure and other diseases

ABSTRACT

The invention relates to a double-stranded compound, preferably an oligoribonucleotide, which down-regulates the expression of a human p53 gene. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the oligoribonucleotide compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating a patient suffering from alopecia or acute renal failure or other diseases comprising administering to the patient the pharmaceutical composition in a therapeutically effective dose so as to thereby treat the patient. The alopecia may be induced by chemotherapy or radiotherapy, and the patient may be suffering from cancer, in particular breast cancer.

This application claims the benefit of U.S. Provisional Application Nos.60/613,991, filed Sep. 28, 2004; 60/658,196, filed Mar. 2, 2005; and60/703,020, filed Jul. 26, 2005, the contents of all of which are herebyincorporated by reference.

Throughout this application various patent and scientific publicationsare cited. The disclosures for these publications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION siRNAs and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Originally,attempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefense mechanism which was activated in response to long dsRNAmolecules; see Gil et al. 2000, Apoptosis, 5:107-114. Later it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without the stimulation of thegeneric antiviral defence mechanisms (see Elbashir et al. Nature 2001,411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747). Asa result, small interfering RNAs (siRNAs), which are shortdouble-stranded RNAs, have become powerful tools in attempting tounderstand gene function.

Thus, RNA interference (RNAi) refers to the process of sequence-specificpost-transcriptional gene silencing in mammals mediated by smallinterfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) ormicroRNAs (miRNAs) (Ambros V. Nature 431:7006, 350-355(2004); and BartelD P. Cell. 2004 Jan. 23; 116(2): 281-97 MicroRNAs: genomics, biogenesis,mechanism, and function). The corresponding process in plants iscommonly referred to as specific post-transcriptional gene silencing orRNA silencing and is also referred to as quelling in fungi. An siRNA isa double-stranded RNA molecule which down-regulates or silences(prevents) the expression of a gene/mRNA of its endogenous (cellular)counterpart. RNA interference is based on the ability of dsRNA speciesto enter a specific protein complex, where it is then targeted to thecomplementary cellular RNA and specifically degrades it. Thus, the RNAinterference response features an endonuclease complex containing ansiRNA, commonly referred to as an RNA-induced silencing complex (RISC),which mediates cleavage of single-stranded RNA having a sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA may take place in the middle of the region complementaryto the antisense strand of the siRNA duplex (Elbashir et al 2001, GenesDev., 15, 188). In more detail, longer dsRNAs are digested into short(17-29 bp) dsRNA fragments (also referred to as short inhibitoryRNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein etal., Nature, 2001, v.409, p. 363-6; Lee et al., Nature, 2003, 425, p.415-9). The RISC protein complex recognizes these fragments andcomplementary mRNA. The whole process is culminated by endonucleasecleavage of target mRNA (McManus&Sharp, Nature Rev Genet, 2002, v.3, p.737-47; Paddison &Hannon, Curr Opin Mol Ther. 2003 June; 5(3): 217-24).For information on these terms and proposed mechanisms, see BernsteinE., Denli A M. Hannon G J: 2001 The rest is silence. RNA. I; 7(11):1509-21; Nishikura K.: 2001 A short primer on RNAi: RNA-directed RNApolymerase acts as a key catalyst. Cell. I 16; 107(4): 415-8 and PCTpublication WO 01/36646 (Glover et al).

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; see for example Chalk A M, Wahlestedt C,Sonnhammer E L. 2004 Improved and automated prediction of effectivesiRNA Biochem. Biophys. Res. Commun. June 18; 319(1): 264-74; Sioud M,Leirdal M., 2004, Potential design rules and enzymatic synthesis ofsiRNAs, Methods Mol Biol.; 252:457-69; Levenkova N, Gu Q, Rux J J. 2004,Gene specific siRNA selector Bioinformatics. I 12; 20(3): 430-2. andUi-Tai K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A,Ueda R, Saigo K., Guidelines for the selection of highly effective siRNAsequences for mammalian and chick RNA interference Nucleic Acids Res.2004 I 9; 32(3):936-48. Se also Liu Y, Braasch D A, Nulf C J, Corey D R.Efficient and isoform-selective inhibition of cellular gene expressionby peptide nucleic acids, Biochemistry, 2004 124; 43(7):1921-7. See alsoPCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al),and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemicalmodification analysis, RNA 2003 September; 9(9):1034-48 and I U.S. Pat.Nos. 5,898,031 and 6,107,094 (Crooke) for production of modified/morestable siRNAs.

Several groups have described the development of DNA-based vectorscapable of generating siRNA within cells. The method generally involvestranscription of short hairpin RNAs that are efficiently processed toform siRNAs within cells. Paddison et al. PNAS 2002, 99:1443-1448;Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002,8:5515-5520; and Brummelkamp at al. Science 2002, 296:550-553. Thesereports describe methods to generate siRNAs capable of specificallytargeting numerous endogenously and exogenously expressed genes.

siRNA has recently been successfully used for inhibition in primates;for further details see Tolentino et al., Retina 24(1) February 2004 I132-138.

The p53 Gene and Polypeptide

The human p53 gene is a well-known and highly studied gene. The p53polypeptide plays a key role in cellular stress response mechanisms byconverting a variety of different stimuli, for example DNA damagingconditions, such as gamma-irradiation, deregulation of transcription orreplication, and oncogene transformation, into cell growth arrest orapoptosis (Gottlieb et al, 1996, Biochem. Biophys. Acta. 127, p. 77).The p53 polypeptide is essential for the induction of programmed celldeath or “apoptosis” as a response to such stimuli. Most anti-cancertherapies damage or kill also normal cells that contain native p53,causing severe side effects associated with the damage or death ofhealthy cells. Since such side effects are to a great extent determinedby p53-mediated death of normal cells, the temporary suppression of p53during the acute phase of anti-cancer therapy has been suggested as atherapeutic strategy to avoid these severe toxic events. This wasdescribed in U.S. Pat. No. 6,593,353 and in Komarov P G et al, 1999, Achemical inhibitor of p53 that protects mice from the side effects ofcancer therapy, Science, 285(5434):1651, 1653. p53 has been shown to beinvolved in chemotherapy and radiation-induced alopecia. (Botcharev etal, 2000, p53 is essential for Chemotherapy-induced Hair Loss, CancerResearch, 60, 5002-5006).

Alopecia

Recently there have been dramatic advances in the understanding of themolecules and pathways regulating hair follicle formation and hairgrowth. Chemotherapy disrupts the proliferation of matrix keratinocytesin the growth bulb that produce the hair shaft. This forces hairfollicles to enter a dystrophic regression stage in which the integrityof the hair shaft is compromised and the hair then breaks and falls out.Because more than 90% of scalp follicles are in growth stage at any onetime, these hairs are rapidly lost after chemotherapy, and thus thealopecia is rapid and extensive (George Cotsarelis and Sarah E. Millar,2001, Towards a molecular understanding of hair loss and its treatment,TRENDS in Molecular Medicine Vol. 7 No. 7). Chemotherapy drugs mostlikely to cause hair loss are: Cisplatinum, Cytarabine,Cyclophosphamide, Doxorubicin, Epirubicin, Etoposide, Ifosfamide andVincristine. Radiation induced general alopecia is observed in virtually100% of patients who receive whole brain radiation (WBR), particularlyof 3000 rad and above.

Hair loss is one of the most feared side effects of chemotherapy amongpatients with cancer, even although hair lost following chemotherapydoes eventually re-grow. From the patient's perspective, hair loss(alopecia) ranks second only to nausea as a distressing side effect ofchemotherapy. About 75% of patients describe chemotherapy induced hairloss as equal to or more devastating than the pain caused by cancer.

Thus, although hair disorders are not life threatening, their profoundimpact on social interactions and on the psychological well-being ofpatients is undeniable. The demand for treatments for hair loss fuels amulti-billion dollar industry. Despite this, most currently marketedproducts are ineffective, evidenced by the fact that the FDA hasapproved only two treatments for hair loss. None of the known therapiesor remedies is effective on cancer therapy-induced alopecia.

Acute Renal Failure (ARF).

ARF is a clinical syndrome characterized by rapid deterioration of renalfunction that occurs within days. The principal feature of ARF is anabrupt decline in glomerular filtration rate (GFR), resulting in theretention of nitrogenous wastes (urea, creatinine). In the general worldpopulation 170-200 cases of severe ARF per million population occurannually. To date, there is no specific treatment for established ARF.Several drugs have been found to ameliorate toxic and ischemicexperimental ARF, as manifested by lower serum creatinine levels,reduced histological damage and faster recovery of renal function indifferent animal models. These include anti-oxidants, calcium channelblockers, diuretics, vasoactive substances, growth factors,anti-inflammatory agents and more. However, those drugs that have beenstudied in clinical trials showed no benefit, and their use in clinicalARF has not been approved.

In the majority of hospitalized patients, ARF is caused by acute tubularnecrosis (ATN), which results from ischemic and/or nephrotoxic insults.Renal hypoperfusion is caused by hypovolemic, cardiogenic and septicshock, by administration of vasoconstrictive drugs or renovascularinjury. Nephrotoxins include exogenous toxins such as contrast media andaminoglycosides as well as endogenous toxin such as myoglobin. Recentstudies, however, support that apoptosis in renal tissues is prominentin most human cases of ARF. The principal site of apoptotic cell deathis the distal nephron. During the initial phase of ischemic injury, lossof integrity of the actin cytoskeleton leads to flattening of theepithelium, with loss of the brush border, loss of focal cell contacts,and subsequent disengagement of the cell from the underlying substratum.It has been suggested that apoptotic tubule cell death may be morepredictive of functional changes than necrotic cell death (Komarov etal. Science. 1999 Sep. 10; 285(5434):1733-7); see also (Supavekin et al.Kidney Int. 2003 May; 63(5):1714-24).

In conclusion, currently there are no satisfactory modes of therapy forthe prevention and/or treatment of toxic alopecia and of acute renalfailure, nor are there a satisfactory mode of therapy for many otherdiseases and disorders which are accompanied by an elevated level of p53polypeptide, and there is a need therefore to develop novel compoundsfor this purpose.

SUMMARY OF THE INVENTION

The invention provides novel double stranded oligoribonucleotides thatinhibit the p53 gene. The invention also provides a pharmaceuticalcomposition comprising one or more such oligoribonucleotides, and avector capable of expressing the oligoribonucleotide. The presentinvention also provides a method of treating a patient suffering from adisease in which temporary (reversible) inhibition of p53 activity isbeneficial comprising administering to the patient one or moreoligoribonucleotides typically as a pharmaceutical composition, in atherapeutically effective dose so as to thereby treat the patient. Thepresent invention also contemplates treating other disorders which areaccompanied by an elevated level of p53 polypeptide. Since long-term p53inactivation can significantly increase the risk of cancer, it ispreferred that the inhibition of p53 using the molecules of the presentinvention will be temporary.

In one preferred embodiment, the novel siRNA molecules disclosed hereinmay be used in the treatment of tumors in cases where temporarysuppression of p53 using the p53 siRNA would be beneficial along withconventional chemotherapy (as described herein) or radiotherapy. Forexample, the novel siRNA molecules disclosed herein would protect normalp53-containing cells from chemotherapy or radiotherapy-inducedapoptosis. The novel siRNA molecules disclosed herein may also be usedfor inhibition of p53 expression in specific cancer cell in cases wherep53 inhibition potentiates apoptotic cell death in these cells.Specifically, radiation therapy and chemotherapy may cause severe sideeffects, such as severe damage to the lymphoid and hematopoietic systemand intestinal epithelia, which limit the effectiveness of thesetherapies, and may cause hair loss which causes psychological distress.These side effects are caused by p53-mediated apoptosis. Therefore, toeliminate or reduce adverse side effects associated with cancertreatment, it would be beneficial to induce temporary inhibition of p53activity in normal cells using the siRNA molecules of the presentinvention, thereby protecting normal tissue.

In another preferred embodiment, the novel siRNA molecules disclosedherein may be used in the treatment of acute renal failure (ARF), whichis characterized by rapid deterioration of renal function associatedwith apoptotic cell death in the renal tissue.

The novel siRNA molecules disclosed herein may also be used in otherconditions in which p53 is activated as a consequence of a variety ofstresses associated with injuries such as a burn, hyperthermia, hypoxiaassociated with a blocked blood supply such as in myocardial infraction,stroke, and ischemia. Temporary p53 inhibition using the siRNA moleculesof the present invention can be therapeutically effective in reducing oreliminating p53-dependent neuronal death in the central nervous system,i.e., brain and spinal cord injury, the preservation of tissues andorgans prior to transplanting, preparation of a host for a bone marrowtransplant, and reducing or eliminating neuronal damage during aseizure.

p53 also plays a role in cell aging. In particular, morphological andphysiological alterations of normal tissues associated with aging may berelated to p53 activity. Senescent cells that accumulate in tissues overtime are known to maintain very high levels of p53-dependenttranscription. p53-dependent secretion of growth inhibitors by senescentcells accumulate in aging tissue. Thus, the siRNA molecules disclosedherein may also be used in suppression of tissue aging.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. This figure represents the nucleotide sequence of the human p53gene-SEQ ID NO: 1.

FIG. 2. This figure the amino acid sequence of the human p53polypeptide-SEQ ID NO:2.

FIG. 3. This figure shows Western Blot results demonstrating the effectof various human p53 siRNAs on p53 expression.

FIG. 4. This figure shows Western Blot results demonstrating the effectof various mouse p53 siRNAs on p53 expression.

FIG. 5. This figure shows the level of serum creatinine as an indicationfor acute renal failure in animals that underwent bilateral kidneyarterial clamp and were treated with p53 siRNA compound or a control, asindicated.

FIG. 6. This figure shows the extent of tubular necrosis in renal tissuein animals that underwent bilateral kidney arterial clamp and weretreated with the p53 siRNA compound.

FIG. 7. This figure demonstrates that p53 siRNA treatment down-regulatedthe expression of the pro-apoptotic gene Puma in the corticalcompartment of the kidney in animal subjected to ischemia-reperfusionkidney injury.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to compounds which down-regulateexpression of the p53 gene particularly to novel small interfering RNAs(siRNAs), and to the use of these novel siRNAs in the treatment ofvarious diseases and medical conditions in particular alopecia or acuterenal failure or a disorder accompanied by an elevated level of p53polypeptide. The inventors of the present invention have found that itis beneficial to induce temporary inhibition of p53 in order to treatany of said diseases or disorders. Methods, molecules and compositionswhich inhibit p53 are discussed herein at length, and any of saidmolecules and/or compositions may be beneficially employed in thetreatment of a patient suffering from any of said conditions.

The present invention provides methods and compositions for inhibitingexpression of a target p53 gene in vivo. In general, the method includesadministering oligoribonucleotides, such as small interfering RNAs(i.e., siRNAs) that are targeted to a particular p53 r-RNA and hybridizeto, or interact with, the mRNAs under biological conditions (within thecell), or a nucleic acid material that can produce siRNA in a cell, inan amount sufficient to down-regulate expression of a target gene by anRNA interference mechanism. In particular, the subject method can beused to inhibit expression of the p53 gene for treatment of a disease.

In accordance with the present invention, the siRNA molecules orinhibitors of the p53 gene may be used as drugs to treat variouspathologies in particular alopecia or acute renal failure or otherdisorders accompanied by an elevated level of p53 polypeptide. Sincelong-term p53 inactivation can significantly increase the risk ofcancer, it is preferred that the inhibition of p53 using the moleculesof the present invention be temporary/reversible.

The present invention provides double-stranded oligoribonucleotides(siRNAs), which down-regulate the expression of the p53 gene. An siRNAof the invention is a duplex oligoribonucleotide in which the sensestrand is derived from the mRNA sequence of the p53 gene, and theantisense strand is complementary to the sense strand. In general, somedeviation from the target mRNA sequence is tolerated withoutcompromising the siRNA activity (see e.g. Czauderna et al 2003 NucleicAcids Research 31(11), 2705-2716). An siRNA of the invention inhibitsgene expression on a post-transcriptional level with or withoutdestroying the mRNA. Without being bound by theory, siRNA may target themRNA for specific cleavage and degradation and/or may inhibittranslation from the targeted message.

There are at least four variant p53 polypeptides (see Bourdon et al.Genes Dev. 2005; 19: 2122-2137). The sequence given in FIG. 1 is thenucleotide sequence of gi-8400737. The corresponding polypeptidesequence has 393 amino acids; see FIG. 2. All variants and any othersimilar minor variants are included in the definition of p53 polypeptideand in the definition of the p53 genes encoding them.

As used herein, the term “p53 gene” is defined as any homolog of the p53gene having preferably 90% homology, more preferably 95% homology, andeven more preferably 98% homology to the amino acid encoding region ofSEQ ID NO: 1 or nucleic acid sequences which bind to the p53 gene underconditions of highly stringent hybridization, which are well-known inthe art (for example, see Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and1998.

As used herein, the term “p53”, or “p53 polypeptide” is defined as anyhomolog of the p53 polypeptide having preferably 90% homology, morepreferably 95% homology, and even more preferably 98% homology to SEQ IDNO:2, as either full-length or a fragment or a domain thereof, as amutant or the polypeptide encoded by a spliced variant nucleic acidsequence, as a chimera with other polypeptides, provided that any of theabove has the same or substantially the same biological function as thep53 polypeptide.

Generally, the siRNAs used in the present invention comprise aribonucleic acid comprising a double stranded structure, whereby thedouble-stranded structure comprises a first strand and a second strand,whereby the first strand comprises a first stretch of contiguousnucleotides and whereby said first stretch is at least partiallycomplementary to a target nucleic acid, and the second strand comprisesa second stretch of contiguous nucleotides and whereby said secondstretch is at least partially identical to a target nucleic acid,whereby said first strand and/or said second strand comprises aplurality of groups of modified nucleotides having a modification at the2′-position whereby within the strand each group of modified nucleotidesis flanked on one or both sides by a flanking group of nucleotideswhereby the flanking nucleotides forming the flanking group ofnucleotides is either an unmodified nucleotide or a nucleotide having amodification different from the modification of the modifiednucleotides. Further, said first strand and/or said second strand maycomprise said plurality of modified nucleotides and may comprises saidplurality of groups of modified nucleotides.

The group of modified nucleotides and/or the group of flankingnucleotides may comprise a number of nucleotides whereby the number isselected from the group comprising one nucleotide to 10 nucleotides. Inconnection with any ranges specified herein it is to be understood thateach range discloses any individual integer between the respectivefigures used to define the range including said two figures definingsaid range. In the present case the group thus comprises one nucleotide,two nucleotides, three nucleotides, four nucleotides, five nucleotides,six nucleotides, seven nucleotides, eight nucleotides, nine nucleotidesand ten nucleotides.

The pattern of modified nucleotides of said first strand may be shiftedby one or more nucleotides relative to the pattern of modifiednucleotides of the second strand.

The modifications discussed above may be selected from the groupcomprising amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro, chloro,bromo, CN, CF, imidazole, caboxylate, thioate, C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl. OCF₃, OCN, O-, S-, orN-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃;heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino orsubstituted silyl, as, among others, described in European patents EP O586 520 B1 or EP O 618 925 B1.

The double stranded structure of the siRNA may be blunt ended, on one orboth sides. More specifically, the double stranded structure may beblunt ended on the double stranded structure's side which is defined bythe 5′-end of the first strand and the 3′-end of the second strand, orthe double stranded structure may be blunt ended on the double strandedstructure's side which is defined by at the 3′-end of the first strandand the 5′-end of the second strand.

Additionally, at least one of the two strands may have an overhang of atleast one nucleotide at the 5′-end; the overhang may consist of at leastone deoxyribonucleotide. At least one of the strands may also optionallyhave an overhang of at least one nucleotide at the 3′-end.

The length of the double-stranded structure of the siRNA is typicallyfrom about 17 to 21 and more preferably 18 or 19 bases. Further, thelength of said first strand and/or the length of said second strand mayindependently from each other be selected from the group comprising theranges of from about 15 to about 23 bases, 17 to 21 bases and 18 or 19bases.

Additionally, the complementarily between said first strand and thetarget nucleic acid may be perfect, or the duplex formed between thefirst strand and the target nucleic acid may comprise at least 15nucleotides wherein there is one mismatch or two mismatches between saidfirst strand and the target nucleic acid forming said double-strandedstructure.

In some cases both the first strand and the second strand each compriseat least one group of modified nucleotides and at least one flankinggroup of nucleotides, whereby each group of modified nucleotidescomprises at least one nucleotide and whereby each flanking group ofnucleotides comprising at least one nucleotide with each group ofmodified nucleotides of the first strand being aligned with a flankinggroup of nucleotides on the second strand, whereby the most terminal 5′nucleotide of the first strand is a nucleotide of the group of modifiednucleotides, and the most terminal 3′ nucleotide of the second strand isa nucleotide of the flanking group of nucleotides. Each group ofmodified nucleotides may consist of a single nucleotide and/or eachflanking group of nucleotides may consist of a single nucleotide.

Additionally, it is possible that on the first strand the nucleotideforming the flanking group of nucleotides is an unmodified nucleotidewhich is arranged in a 3′ direction relative to the nucleotide formingthe group of modified nucleotides, and on the second strand thenucleotide forming the group of modified nucleotides is a modifiednucleotide which is arranged in 5′ direction relative to the nucleotideforming the flanking group of nucleotides.

Further the first strand of the siRNA may comprise eight to twelve,preferably nine to eleven, groups of modified nucleotides, and thesecond strand may comprise seven to eleven, preferably eight to ten,groups of modified nucleotides.

The first strand and the second strand may be linked by a loopstructure, which may be comprised of a non-nucleic acid polymer such as,inter alia, polyethylene glycol. Alternatively, the loop structure maybe comprised of a nucleic acid.

Further, the 5′-terminus of the first strand of the siRNA may be linkedto the 3′-terminus of the second strand, or the 3′-end of the firststrand may be linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 10-2000 nucleobases.

In particular, the invention provides a compound having structure A:

5′(N)_(x)—Z3′(antisense strand)

3′Z′—(N′)_(y)5′(sense strand)

-   -   wherein each N and N′ is a ribonucleotide which may be modified        or unmodified in its sugar residue and (N)_(x) and (N′)_(y) is        oligomer in which each consecutive N or N′ is joined to the next        N or N′ by a covalent bond;    -   wherein each of x and y is an integer between 19 and 40;    -   wherein each of Z and Z′ may be present or absent, but if        present is dTdT and is covalently attached at the 3′ terminus of        the strand in which it is present; and wherein the sequence of        (N), comprises an antisense sequence to mRNA of p53 in        particular any of the antisense sequences present in any of        Tables A, B and C.

It will be readily understood by those skilled in the art that thecompounds of the present invention consist of a plurality ofnucleotides, which are linked through covalent linkages. Each suchcovalent linkage may be a phosphodiester linkage, a phosphothioatelinkage, or a combination of both, along the length of the nucleotidesequence of the individual strand. Other possible backbone modificationsare described inter alia in U.S. Pat. Nos. 5,587,361; 6,242,589;6,277,967; 6,326,358; 5,399,676; 5,489,677; and 5,596,086.

In particular embodiments, x and y are preferably an integer betweenabout 19 to about 27, most preferably from about 19 to about 23. In aparticular embodiment of the compound of the invention, x may be equalto y (viz., x=y) and in preferred embodiments x=y=19 or x=y=21. In aparticularly preferred embodiment x=y=19.

In one embodiment of the compound of the invention, Z and Z′ are bothabsent; in another embodiment one of Z or Z′ is present.

In one embodiment of the compound of the invention, all of theribonucleotides of the compound are unmodified in their sugar residues.

In preferred embodiments of the compound of the invention, at least oneribonucleotide is modified in its sugar residue, preferably amodification at the 2′ position. The modification at the 2′ positionresults in the presence of a moiety which is preferably selected fromthe group comprising amino, fluoro, methoxy, alkoxy and alkyl groups. Ina presently most preferred embodiment the moiety at the 2′ position ismethoxy (2′-0-methyl).

In preferred embodiments of the invention, alternating ribonucleotidesare modified in both the antisense and the sense strands of thecompound. In particular the siRNA used in the Examples has been suchmodified such that a 2′ O-Me group was present on the first, third,fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth andnineteenth nucleotide of the antisense strand, whereby the very samemodification, i. e. a 2′-O-Me group was present at the second, fourth,sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenthnucleotide of the sense strand. Additionally, it is to be noted that thein case of these particular nucleic acids according to the presentinvention the first stretch is identical to the first strand and thesecond stretch is identical to the second strand and these nucleic acidsare also blunt ended.

In a particularly preferred embodiment the sequence of the siRNA is thatof 15 in Table A.

According to one preferred embodiment of the invention, the antisenseand the sense strands of the siRNA molecule are both phophorylated onlyat the 3′-terminus and not at the 5′-terminus. According to anotherpreferred embodiment of the invention, the antisense and the sensestrands are both non-phophorylated both at the 3′-terminus and also atthe 5′-terminus. According to yet another preferred embodiment of theinvention, the 1^(st) nucleotide in the 5′ position in the sense strandis specifically modified to abolish any possibility of in vivo5′-phosphorylation.

In another embodiment of the compound of the invention, theribonucleotides at the 5′ and 3′ termini of the antisense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the sense strand are unmodified in their sugar residues.

The invention farther provides a vector capable of expressing any of theaforementioned oligoribonucleotides in unmodified form in a cell afterwhich appropriate modification may be made.

The invention also provides a composition comprising one or more of thecompounds of the invention in a carrier, preferably a pharmaceuticallyacceptable carrier. This composition may comprise a mixture of two ormore different siRNAs.

The invention also provides a composition which comprises the abovecompound of the invention covalently or non-covalently bound to one ormore compounds of the invention in an amount effective to inhibit humanp53 and a carrier. This composition may be processed intracellularly byendogenous cellular complexes to produce one or moreoligoribonucleotides of the invention.

The invention also provides a composition comprising a carrier and oneor more of the compounds of the invention in an amount effective todown-regulate expression in a cell of a human p53, which compoundcomprises a sequence substantially complementary to the sequence of(N)_(x).

Additionally the invention provides a method of down-regulating theexpression of gene p53 by at least 50% as compared to a controlcomprising contacting an mRNA transcript of gene p53 with one or more ofthe compounds of the invention.

In one embodiment the oligoribonucleotide is down-regulating p53,whereby the down-regulation of p53 is selected from the group comprisingdown-regulation of p53 function, down-regulation of p53 polypeptide anddown-regulation of p53 mRNA expression.

In one embodiment the compound is down-regulating a p53 polypeptide,whereby the down-regulation of p53 is selected from the group comprisingdown-regulation of p53 function (which may be examined by an enzymaticassay or a binding assay with a known interactor of the nativegene/polypeptide, inter alia), down-regulation of p53 protein (which maybe examined by Western blotting, ELISA or immuno-precipitation, interalia) and down-regulation of p53 mRNA expression (which may be examinedby Northern blotting, quantitative RT-PCR, in-situ hybridisation ormicroarray hybridisation, inter alia).

The invention also provides a method of treating a patient sufferingfrom a disease accompanied by an elevated level of p53 polypeptide, themethod comprising administering to the patient a composition of theinvention in a therapeutically effective dose thereby treating thepatient. Preferably, the present invention provides a method of treatinga patient suffering from a disease in which temporary inhibition of p53is beneficial. In one preferred embodiment, the compositions of thepresent invention are used for the treatment of tumors along with theconventional chemotherapy or radiotherapy in order to prevent thealopecia associated with chemotherapy or radiotherapy. In anotherpreferred embodiment, the compositions of the present invention are usedfor the treatment of acute renal failure. In yet another preferredembodiment, the compositions of the present invention are used inconditions in which p53 is activated as a consequence of a variety ofstresses associated with injuries such as a burn, hyperthermia, hypoxiaassociated with a blocked blood supply such as in myocardial infraction,stroke, and ischemia. Temporary p53 inhibition using the siRNA moleculesof the present invention can be therapeutically effective in reducing oreliminating p53-dependent neuronal death in the central nervous system,i.e., brain and spinal cord injury, in preserving of tissue and an organprior to transplanting preparation of a host for a bone marrowtransplant, reducing or eliminating neuronal damage during a seizure andin suppressing tissue aging.

The invention also provides a use of a therapeutically effective dose ofone or more compounds of the invention for the preparation of acomposition for the treatment of a disease accompanied by an elevatedlevel of p53 polypeptide, such as in a patient suffering from alopeciaor acute renal failure.

More particularly, the invention provides an oligoribonucleotide whereinone strand comprises consecutive nucleotides having, from 5′ to 3′, thesequence set forth in SEQ ID NOS: 3-25 (Table A, sense strands) or inSEQ ID NOS: 49-119 (Table B, sense strands) or in SEQ ID NOS: 191-253(Table C, sense strands) or a homolog thereof wherein in up to 2 of thenucleotides in each terminal region a base is altered.

The terminal region of the oligonucleotide refers to bases 1-4 and/or16-19 in the 19-mer sequence and to bases 1-4 and/or 18-21 in the 21-mersequence.

Additionally, the invention provides oligoribonucleotides wherein onestrand comprises consecutive nucleotides having, from 5′ to 3′, thesequence set forth SEQ ID NOS: 26-48 (Table A, antisense strands) or SEQID NOS: 120-190 (Table B, antisense strands) or SEQ ID NOS: 254-316(Table C, antisense strands) or a homolog thereof wherein in up to 2 ofthe nucleotides in each terminal region a base is altered.

The preferred oligonucleotides of the invention are human p53oligonucleotides serial numbers 3, 5, 20 and 23 in Table D and mouse p53oligonucleotides serial numbers 1 11, 12, 14, 17 and 18 in Table E.These are identical to serial numbers 3, 5, 20 and 23 (human) and also11, 12, 14, 17 and 18 (mouse) in Table A. The most preferredoligonucleotides of the invention are human p53 oligonucleotides havingthe sequence of serial number 23 in Table A.

The presently most preferred compound of the invention is a blunt-ended19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent. Theoligonucleotide molecule is either phosphorylated at 3′ termini of bothsense and anti-sense strands, or non-phosphorylated at all; or having1^(st) nucleotide in the 5′ position on the sense strand specificallymodified to abolish any possibility of in vivo 5′-phosphorylation. Thealternating ribonucleotides are modified at the 2′ position in both theantisense and the sense strands, wherein the moiety at the 2′ positionis methoxy (2′-0-methyl) and wherein the ribonucleotides at the 5′ and3′ termini of the antisense strand are modified in their sugar residues,and the ribonucleotides at the 5′ and 3′ termini of the sense strand areunmodified in their sugar residues. The presently most preferred suchcompounds are such modified oligonucleotides comprising the sequenceshaving serial number 23 in Table A.

In one aspect of the invention the oligonucleotide comprises adouble-stranded structure, whereby such double-stranded structurecomprises

-   -   a first strand and a second strand, whereby    -   the first strand comprises a first stretch of contiguous        nucleotides and the second strand comprises a second stretch of        contiguous nucleotides, whereby    -   the first stretch is either complementary or identical to a        nucleic acid sequence coding for p53 and whereby the second        stretch is either identical or complementary to a nucleic acid        sequence coding for p53.

In an embodiment the first stretch and for the second stretch comprisesfrom about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides,more preferably from about 19 to 27 nucleotides and most preferably fromabout 19 to 23 nucleotides, in particular from about 19 to 21nucleotides. In such an embodiment the oligonucleotide may be from 17-40nucleotides in length.

Additionally, further nucleic acids according to the present inventioncomprise at least 14 contiguous nucleotides of any one of thepolynucleotides in the Tables and more preferably 14 contiguousnucleotide base pairs at any end of the double-stranded structurecomprised of the first stretch and second stretch as described above.

In an embodiment the first stretch comprises a sequence of at least 14contiguous nucleotides of an oligonucleotide, whereby sucholigonucleotide is selected from the group comprising SEQ. ID. Nos3-316, preferably from the group comprising the oligoribonucleotides ofhaving the sequence of any of the serial numbers 3, 5, 20 or 23 (human)or having the sequence of any of the serial numbers it, 12, 14, 17 and18 (mouse) in Table A, more preferably selected from the group havingthe sequence of any of the serial numbers 3, 5, 20 or 23 in Table A.

Additionally, further nucleic acids according to the present inventioncomprise at least 14 contiguous nucleotides of any one of the SEQ. ID.NO. 3 to 316, and more preferably 14 contiguous nucleotide base pairs atany end of the double-stranded structure comprised of the first stretchand second stretch as described above. It will be understood by oneskilled in the art that given the potential length of the nucleic acidaccording to the present invention and particularly of the individualstretches forming such nucleic acid according to the present invention,some shifts relative to the coding sequence of p53 to each side ispossible, whereby such shifts can be up to 1, 2, 3, 4, 5 and 6nucleotides in both directions, and whereby the thus generateddouble-stranded nucleic acid molecules shall also be within the presentinvention.

Delivery:

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, see, forexample, Shen et al (FEBS letters 539: 111-114 (2003)), Xia et al.,Nature Biotechnology 20: 1006-1010 (2002), Reich et al., MolecularVision 9: 210-216 (2003), Sorensen et al. (J. Mol. Biol. 327: 761-766(2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni etal., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recentlybeen successfully used for inhibition in primates; for further detailssee Tolentino et al., Retina 24(1) February 2004 I 132-138. Respiratoryformulations for siRNA are described in U.S. patent application No.2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and othersteroid and lipid conjugated siRNAs) can been used for delivery seeSoutschek et al Nature 432: 173-177(2004) Therapeutic silencing of anendogenous gene by systemic administration of modified siRNAs; andLorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004) Steroidand lipid conjugates of siRNAs to enhance cellular uptake and genesilencing in liver cells.

The siRNAs or pharmaceutical compositions of the present invention areadministered and dosed in accordance with good medical practice, takinginto account the clinical condition of the individual patient, thedisease to be treated, the site and method of administration, schedulingof administration, patient age, sex, body weight and other factors knownto medical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. The compounds of thepresent invention can be administered by any of the conventional routesof administration. It should be noted that the compound can beadministered as the compound or as pharmaceutically acceptable salt andcan be administered alone or as an active ingredient in combination withpharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. Liquid forms may be prepared for injection, the termincluding subcutaneous, transdermal, intravenous, intramuscular,intrathecal, and other parental routes of administration. The liquidcompositions include aqueous solutions, with and without organicco-solvents, aqueous or oil suspensions, emulsions with edible oils, aswell as similar pharmaceutical vehicles. In addition, under certaincircumstances the compositions for use in the novel treatments of thepresent invention may be formed as aerosols, for intranasal and likeadministration. The patient being treated is a warm-blooded animal and,in particular, mammals including man. The pharmaceutically acceptablecarriers, solvents, diluents, excipients, adjuvants and vehicles as wellas implant carriers generally refer to inert, non-toxic solid or liquidfillers, diluents or encapsulating material not reacting with the activeingredients of the invention and they include liposomes andmicrospheres. Examples of delivery systems useful in the presentinvention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;4,439,196; and 4,475,196. Many other such implants, delivery systems,and modules are well known to those skilled in the art. In one specificembodiment of this invention topical and transdermal formulations areparticularly preferred.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-4weeks or longer.

It is noted that the delivery of the siRNA compounds according to thepresent invention to the target cells in the kidney proximal tubules isparticularly effective in the treatment of acute renal failure. Withoutbeing bound by theory, this may be due to the fact that normally siRNAmolecules are excreted from the body via the cells of the kidneyproximal tubules. Thus, naked siRNA molecules are naturally concentratedin the cells that are targeted for the therapy in acute renal failure.

The term “treatment” as used herein refers to administration of atherapeutic substance effective to ameliorate symptoms associated with adisease, to lessen the severity or cure the disease, or to prevent thedisease from occurring.

In a particular embodiment, the administration comprises intravenousadministration. In another particular embodiment the administrationcomprises topical or local administration

Another aspect of the invention is a method of treating a patientsuffering from alopecia or acute renal failure or a disorder which isaccompanied by an elevated level of p53 polypeptide, comprisingadministering to the patient a pharmaceutical composition of theinvention in a therapeutically effective amount so as to thereby treatthe patient.

In a preferred embodiment for treatment of alopecia, the administrationcomprises topical or local administration. In another preferredembodiment the administration comprises transdermal administration. In aparticular embodiment the pharmaceutical composition is applied to thescalp of the patient. In a preferred embodiment for treatment of ARF,the administration comprises intravenous, intra-arterial orintra-peritoneal administration

Another aspect of the invention is a method of preventing alopecia in apatient undergoing treatment which causes alopecia, comprisingadministering to the patient a pharmaceutical composition of theinvention in a therapeutically effective amount so as to thereby treatthe patient.

In another aspect of the invention a pharmaceutical composition isprovided which comprises any of the above oligoribonucleotides (SEQ IDNOS: 3-316) or vectors and a pharmaceutically acceptable carrier.Another aspect of the invention is the use of a therapeuticallyeffective amount of any of the above oligoribonucleotides (SEQ ID NOS:3-316) or vectors for the preparation of a medicament for promotingrecovery in a patient suffering from alopecia or acute renal failure ora disorder which is accompanied by an elevated level of p53.

In a preferred embodiment, the medicament comprises a topicalmedicament. In a particular embodiment the medicament is applied to thescalp of the patient. In another preferred embodiment the medicamentcomprises transdermal administration

In all the above aspects of the invention the alopecia may be induced bychemotherapy or by radiotherapy and is then termed “toxic alopecia”. Inmore detail, toxic alopecia may be caused by irradiation such as gammairradiation or by chemotherapeutic agents such as etoposide, 5-FU(5-fluorouracil), cis-platinum, doxorubicin, a vinca alkaloid,vincristine, vinblastine, vinorelbine, taxol, cyclophosphamide,ifosfamide, chlorambucil, busulfan, mechlorethamine, mitomycin,dacarbazine, carboplatinum, thiotepa, daunorubicin, idarubicin,mitoxantrone, bleomycin, esperamicin A1, dactinomycin, plicamycin,carmustine, lomustine, tauromustine, streptozocin, melphalan,dactinomycin, procarbazine, dexamethasone, prednisone,2-chlorodeoxyadenosine, cytarabine, docetaxel, fludarabine, gemcitabine,herceptin, hydroxyurea, irinotecan, methotrexate, oxaliplatin, rituxin,semustine, epinrbicin, etoposide, tomudex and topotecan, or a chemicalanalog of one of these chemotherapeutic agents. The chemotherapeuticagents most likely to cause hair loss are: cis-platinum, cytarabine,cyclophosphamide, doxorubicin, epirubicin, etoposide, ifosfamide andvincristine.

The compounds of the invention are preferably used for treating acuterenal failure, in particular acute renal failure due to ischemia in postsurgical patients, and acute renal failure due to chemotherapy treatmentsuch as cisplatin administration or sepsis-associated acute renalfailure. A preferred use of the compounds of the invention is for theprevention of acute renal failure in high-risk patients undergoing majorcardiac surgery or vascular surgery. The patients at high-risk ofdeveloping acute renal failure can be identified using various scoringmethods such as the Cleveland Clinic algorithm or that developed by USAcademic Hospitals (QMMI) and by Veterans' Administration (CICSS). Otherpreferred uses of the compounds of the invention are for the preventionof ischemic acute renal failure in kidney transplant patients or for theprevention of toxic acute renal failure in patients receivingchemotherapy. Other uses are for wound healing, acute liver failure,cisplatin-induced deafness (perhaps topically), ex vivo expansion ofhematopoietic stem cells, preservation of donor organs/tissues beforetransplantation by soaking in siRNA solution (perhaps byelectroporation) and subsequent improvement of graft tissue survivalfollowing transplantation. Other indications may be stroke, Parkinson'sdisease, Alzheimer's disease, doxorubicin-induced cardiotoxicity,myocardial infarction/heart failure and improvement of graft tissuesurvival following transplantation (by systemic administration). Withoutbeing bound by theory all these disorders are accompanied by an elevatedlevel of p53 polypeptide.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises:

-   -   obtaining one or more double stranded compound of the invention;        and    -   admixing said compound with a pharmaceutically acceptable        carrier.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises admixing one or morecompounds of the present invention with a pharmaceutically acceptablecarrier.

In a preferred embodiment, the compound used in the preparation of apharmaceutical composition is admixed with a carrier in apharmaceutically effective dose. In a particular embodiment the compoundof the present invention is conjugated to a steroid or to a lipid or toanother suitable molecule e.g. to cholesterol.

Modifications or analogs of nucleotides can be introduced to improve thetherapeutic properties of the nucleotides. Improved properties includeincreased nuclease resistance and/or increased ability to permeate cellmembranes.

Accordingly, the present invention also includes all analogs of, ormodifications to, a oligonucleotide of the invention that does notsubstantially affect the function of the polynucleotide oroligonucleotide. In a preferred embodiment such modification is relatedto the base moiety of the nucleotide, to the sugar moiety of thenucleotide and/or to the phosphate moiety of the nucleotide.

In embodiments of the invention, the nucleotides can be selected fromnaturally occurring or synthetically modified bases. Naturally occurringbases include adenine, guanine, cytosine, thymine and uracil. Modifiedbases of the oligonucleotides include inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl-adenines, 5-halouracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudouracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine;8-thiolalkyl adenine, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanine, 8-hydroxyl guanine and other substituted guanines, other azaand deaza adenines, other aza and deaza guanines, 5-trifluoromethyluracil and 5-trifluoro cytosine.

In addition, analogs of nucleotides can be prepared wherein thestructures of the nucleotides are fundamentally altered and are bettersuited as therapeutic or experimental reagents. An example of anucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replacedwith a polyamide backbone similar to that found in peptides. PNA analogshave been shown to be resistant to degradation by enzymes and to haveextended lives in vivo and in vitro. Further, PNAs have been shown tobind more strongly to a complementary DNA sequence than to a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, cyclic backbones,or acyclic backbones.

In one embodiment the modification is a modification of the phosphatemoiety, whereby the modified phosphate moiety is selected from the groupcomprising phosphothioate.

The compounds of the present invention can be synthesized by any of themethods that are well-known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotideas. Such synthesis is, among others,described in Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48:2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49:6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154:287-313; the synthesis of thioates is, among others, described inEckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNAmolecules is described in Sproat B., in Humana Press 2005 edited byHerdewijn P.; Kap. 2: 17-31 and respective downstream processes are,among others, described in Pingoud A. et. al., in IRL Press 1989 editedby Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005edited by Herdewijn P.; Kap. 2: 17-31 (supra).

Other synthetic procedures are known in the art e.g. the procedures asdescribed in Usman et al., 1987. J. Am. Chem. Soc., 109, 7845; Scaringeet al., 1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684; and Wincott et al., 1997. Methods Mol.Bio., 74, 59, and these procedures may make use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoranidites at the 3′-end. The modified (e.g. 2′-O-methylated)nucleotides and unmodified nucleotides are incorporated as desired.

The oligonucleotides of the present invention can be synthesizedseparately and joined together post-synthetically, for example, byligation (Moore et al., 1992. Science 256, 9923; Draper et al.,International PCT publication No. WO93/23569; Shabarova et al., 1991.Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides &Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

It is noted that a commercially available machine (available, interalia, from Applied Biosystems) can be used; the oligonucleotides amprepared according to the sequences disclosed herein. Overlapping pairsof chemically synthesized fragments can be ligated using methods wellknown in the art (e.g., see U.S. Pat. No. 6,121,426). The strands amsynthesized separately and then are annealed to each other in the tube.Then, the double-stranded siRNAs are separated from the single-strandedoligonucleotides that were not annealed (e.g. because of the excess ofone of them) by HPLC. In relation to the siRNAs or siRNA fragments ofthe present invention, two or more such sequences can be synthesized andlinked together for use in the present invention.

The compounds of the invention can also be synthesized via a tandemsynthesis methodology, as described in US patent application publicationNo. US2004/0019001 (McSwiggen), wherein both siRNA strands aresynthesized as a single contiguous oligonucleotide fragment or strandseparated by a cleavable linker which is subsequently cleaved to provideseparate siRNA fragments or strands that hybridize and permitpurification of the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker.

The present invention further provides for a pharmaceutical compositioncomprising two or more siRNA molecules for the treatment of any of thediseases and conditions mentioned herein, whereby said two molecules maybe physically mixed together in the pharmaceutical composition inamounts which generate equal or otherwise beneficial activity, or may becovalently or non-covalently bound, or joined together by a nucleic acidlinker of a length ranging from 2-100, preferably 2-50 or 2-30nucleotides. In one embodiment, the siRNA molecules are comprised of adouble-stranded nucleic acid structure as described herein, wherein thetwo siRNA sequences are selected from Tables A-C, preferably from TableA, ID Nos: 3, 5, 20 and 23 (human sequences) and 11, 12, 14, 17 and 18(mouse sequences).

In another embodiment, the siRNA molecules are comprised of adouble-stranded nucleic acid structure, wherein the first siRNA sequenceis selected from Tables A-C, preferably from Table A, ID Nos: 3, 5, 20and 23 (human p53 sequences) or 11, 12, 14, 17 and 18 (mouse p53sequences) and the second siRNA molecule targets a pro-apoptotic gene,thereby providing beneficial activity. The tandem double-strandedstructure which comprises two or more siRNA sequences is processedintracellularly to form two or more different siRNAs. Such second siRNAmolecule is preferably an siRNA molecule that targets a pro-apoptoticgene.

The siRNA molecules are covalently or non-covalently bound or joined bya linker to form a tandem siRNA molecule. Such tandem siRNA moleculescomprising two siRNA sequences are typically of 38-150 nucleotides inlength, more preferably 38 or 40-60 nucleotides in length, and longeraccordingly if more than two siRNA sequences are included in the tandemmolecule. A longer tandem molecule comprised of two or more longersequences which encode siRNA produced via internal cellular processing,e.g., long dsRNAs, is also envisaged, as is a tandem molecule encodingtwo or more shRNAs. Such tandem molecules are also considered to be apart of the present invention.

siRNA molecules that target p53 may be the main active component in apharmaceutical composition, or may be one active component of apharmaceutical composition containing two or more siRNAs (or moleculeswhich encode or endogenously produce two or more siRNAs, be it a mixtureof molecules or one or more tandem molecules which encode two or moresiRNAs), said pharmaceutical composition further being comprised of oneor more additional siRNA molecule which targets one or more additionalgene. Simultaneous inhibition of p53 and said additional gene(s) willlikely have an additive or synergistic effect for treatment of thediseases disclosed herein.

In a specific example, the pharmaceutical composition for treatment ofthe diseases disclosed herein may be comprised of the following compoundcombinations: 1) p53 siRNA and Fas siRNA; 2) p53 siRNA and Bax siRNA; 3)p53 siRNA and Noxa siRNA; 4) p53 siRNA and Puma siRNA; 5) p53 siRNA andRTP801 siRNA; 6) p53 siRNA and PIDD siRNA; 7) p53 siRNA, Fas siRNA andany of RTP801 siRNA, Bax siRNA, Noxa siRNA or Puma siRNA or PIDD siRNAto form trimers or polymers (i.e., tandem molecules which encode threesiRNAs). Other preferred options of pro-apoptotic genes to be combinedwith the p53 siRNA are TNFα, caspase 2, caspase 3, caspase 9, E2F1, andPARP-1. A preferred combination according to the present invention isp53 siRNA and RTP801 siRNA. (see PCT patent application PCT/EP2005/008891).

Additionally, p53 siRNA or any nucleic acid molecule comprising orencoding p53 siRNA can be linked (covalently or non-covalently) toantibodies against cell surface internalizable molecules expressed onthe target cells, in order to achieve enhanced targeting for treatmentof the diseases disclosed herein. For example, anti-Fas antibody(preferably a neutralizing antibody) may be combined with a p53 siRNAmolecule.

The compounds of the present invention can be delivered either directlyor with viral or non-viral vectors. When delivered directly thesequences are generally rendered nuclease resistant. Alternatively thesequences can be incorporated into expression cassettes or constructssuch that the sequence is expressed in the cell as discussed hereinbelow. Generally the construct contains the proper regulatory sequenceor promoter to allow the sequence to be expressed in the targeted cell.Vectors optionally used for delivery of the compounds of the presentinvention are commercially available, and may be modified for thepurpose of delivery of the compounds of the present invention by methodsknown to one of skill in the art.

It is also envisaged that a long oligonucleotide (typically 25-500nucleotides in length) comprising one or more stem and loop structures,where stem regions comprise the sequences of the oligonucleotides of theinvention, may be delivered in a carrier, preferably a pharmaceuticallyacceptable carrier, and may be processed intracellularly by endogenouscellular complexes (e.g. by DROSHA and DICER as described above) toproduce one or more smaller double stranded oligonucleotides (siRNAs)which are oligonucleotides of the invention. This oligonucleotide can betermed a tandem shRNA construct. It is envisaged that this longoligonucleotide is a single stranded oligonucleotide comprising one ormore stem and loop structures, wherein each stem region comprises asense and corresponding antisense siRNA sequence of an p53 gene. Inparticular, it is envisaged that this oligonucleotide comprises senseand antisense siRNA sequences as depicted in any one of Tables A, B orC.

As used herein, the term “polypeptide” refers to, in addition to apolypeptide, an oligopeptide, peptide and a full protein.

Screening of p53 Inactivation Compounds:

Some of the compounds and compositions of the present invention may beused in a screening assay for identifying and isolating compounds thatmodulate the activity of p53, in particular compounds that modulatealopecia or acute renal failure or a disorder accompanied by an elevatedlevel of p53 polypeptide. The compounds to be screened comprise interalia substances such as small chemical molecules and antisenseoligonucleotides.

The inhibitory activity of the compounds of the present invention on p53polypeptide activity or binding of the compounds of the presentinvention to p53 may be used to determine the interaction of anadditional compound with the p53 polypeptide, e.g., if the additionalcompound competes with the oligonucleotides of the present invention forp53 inhibition, or if the additional compound rescues said inhibition.The inhibition or activation can be tested by various means, such as,inter alia, assaying for the product of the activity of the p53polypeptide or displacement of binding compound from the p53 polypeptidein radioactive or fluorescent competition assays.

The present invention is illustrated in detail below with reference toExamples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES General Methods in Molecular Biology

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Bitten et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) wascarried out generally as in PCR Protocols: A Guide To Methods AndApplications, Academic Press, San Diego, Calif. (1990). In situ (Incell) PCR in combination with Flow Cytometry can be used for detectionof cells containing specific DNA and mRNA sequences (Testoni et al.,1996, Blood 87:3822.) Methods of performing RT-PCR are also well knownin the art.

Example 1 Generation of Sequences for Active siRNA Compounds

Using proprietary algorithms and the known sequence of gene p53 (SEQ IDNO:1), the sequences of many potential siRNAs were generated. Table Ashows 23 siRNAs which have so far been selected, chemically synthesizedand tested for activity (see Example 2). All these siRNAs are 19-mers.

TABLE A Sense Antisense NM_000546 NM_011640 NM_030989 Number Indexstrand strand Species (human) (mouse) (rat)  1 Mo3 GUACAUGUGU GGAGCUAUUAmouse 3 mis 1232-1250 2 mis AAUAGCUCC CACAUGUAC  2 Hu2 GACUCCAGUGGUAGAUUACC human* 1026-1044 3 mis 2 mis GUAAUCUAC ACUGGAGUC  3 QHMon1CAGACCUAUG AGUAGUUUCC hum, mon 310-328 3 mis 4 mis GAAACUACU AUAGGUCUG 4 QHMon2 CUACCUCCCG UUUUUAUGGC hum, mon 1378-1396 1 mis 1 mis CCAUAAAAAGGGAGGUAG  5 QH1 CCCAAGCAAU AAAUCAUCCA human 361-379 No No GGAUGAUUUUUGCUUGGG  6 QH2 CCCGGACGAU UUGUUCAAUA human 389-407 No No AUUGAACAAUCGUCCGGG  7 QM1 GAGUCACAGU UGAUAUCCGA mouse No 552-578 2 mis CGGAUAUCACUGUGACUC  8 QM2 GGAUGUUGAG AAAAAACUCC mouse No 680-698 4 mis GAGUUUUUUUCAACAUCC  9 QM3 CAUCUUUUGU UGAGAAGGGA mouse 2 mis 808-826 2 misCCCUUCUCA CAAAAGAUG 10 QM6 GGAAUAGGUU ACAACUAUCA mouse No 1870-1888 NoGAUAGUUGU ACCUAUUCC 11 QM4 GGACAGCCAA AUAACAGACU mouse, rat 2 mis877-895 527-545 GUCUGUUAU UGGCUGUCC 12 QM5 GAAGAAAAUU UUUUGCGGAAmouse, rat 3 mis 1383-1401 1033-1051 UCCGCAAAA AUUUUCUUC 13 A17CUGGGACAGC ACAGACUUGG hum, mus 598-616 874-892 2 mis CAAGUCUGU CUGUCCCAG14 E2 UCAUCACACU GAGUCUUCCA hum, mus, 1012-1030 1288-1306 938-956GGAAGACUC GUGUGAUGA rat 15 E6 CACACUGGAA ACUGGAGUCU hum, mus, 1016-10341292-1310 942-960 GACUCCAGU UCCAGUGUG rat 16 B1 GCGCCAUGGC UUGUAGAUGGhum, mon, 724-742 1000-1018 652-668 CAUCUACAA CCAUGGCGC mus (17) 17 B2CGCCAUGGCC CUUGUAGAUG hum, mon, 725-743 1001-1019 652-669 AUCUACAAGGCCAUGGCG mus (18) 18 C1 AGUCACAGCA UCCGUCAUGU hum, mon, 745-7631021-1039 2 mis CAUGACGGA GCUGUGACU mus 19 F2 UCCGAGUGGA AAAUUUCCUUhum, mon, 835-853 1 mis 3 mis AGGAAAUUU CCACUCGGA dog 20 F3 CCGAGUGGAACAAAUUUCCU hum, mon, 836-854 1 mis 3 mis GGAAAUUUG UCCACUCGG dog 21 G1GACAGAAACA GUCGAAAAGU hum, mon, 873-891 No No CUUUUCGAC GUUUCUGUC dog 22H2 GUGUGGUGGU UCAUAGGGCA hum, mon, 895-913 3 mis 3 mis GCCCUAUGACCACCACAC dog 23 I5 GAGAAUAUUU UGAAGGGUGA hum, mon, 1225-1243 2 mis1 mis CACCCUUCA AAUAUUCUC dog

Note that in the above Table A, the sense strands of siRNAs 1-23 haveSEQ ID NOS: 3-25 respectively, and the antisense strands of siRNAs 1-23have SEQ ID NOS: 26-48 respectively. siRNA compound No 1 (SEQ ID NOS: 3and 26) is known from the literature (Dirac and Bernards, Reversal ofsenescence in mouse fibroblasts through lentiviral suppression of p53,J. Biol. Chem. (2003) 278:11731) and siRNA No 2 (SEQ ID NOS:4 and 27) isalso known from the literature (Brummelkamp et al. Science 2002,296:550-553). However, the use of these compounds in the methods oftreatment disclosed herein is previously undisclosed and thus novel.

Table B below shows 71 additional 19-mer sRNAs which have been generatedby the proprietary algorithms.

TABLE B gi2689466 gi5357emb gbU48957.1 X01237 gi4996229dbj gi13097806U48957 JMMP53R AB020761.1 gbBC003596.1 (Macaca (Mouse (Canis No. SourceSense AntiSense (Homo sapiens) fascicularis) mRNA) familiaris)  1 HumanGUACCACCAU UUGUAGUGGA [806-824] [835-852] CCACUACAA UGGUGGUAC  2 HumanGGAAACUACU UUUUCAGGAA [188-206] [234-247] UCCUGAAAA CUAGUUUCC  3 HumanAGACUCCAGU UAGAUUACCA [894-912] [922-933] GGUAAUCUA CUGGAGUCU  4 HumanCCAUCCACUA AUGUAGUUGU [812-830] [840-858] CAACUACAU AGUGGAUGG  5 HumanCCACCAUCCA UAGUUGUAGU [809-827] [837-852] CUACAACUA GGAUGGUGG  6 HumanAAACACUUUU ACUAUGUCGA [747-765] — CGACAUAGU AAAGUGUUU  7 HumanCAUGAGCGCU UAUCUGAGCA [655-673] [683-696] GCUCAGAUA GCGCUCAUG  8 HumanCCAUGGCCAU UGCUUGUAGA [596-614] [624-640] CUACAAGCA UGGCCAUGG  9 HumanCCAAGUCUGU GUGCAAGUCA [476-494] — GACUUGCAC CAGACUUGG 10 HumanAAACUUUGCU UUUUUUGGCA [2476-2494] — GCCAAAAAA GCAAAGUUU 11 HumanCCCUCCUUCU UAAAAAGGGA [2421-2439] — CCCUUUUUA GAAGGAGGG 12 HumanGCAAGCACAU AAAAUGCAGA [2389-2407] — CUGCAUUUU UGUGCUUGC 13 HumanGGGUCAACAU AUGUAAAAGA [2367-2385] — CUUUUACAU UGUUGACCC 14 HumanGAAGGGUCAA UAAAAGAUGU [2364-2382] — CAUCUUUUA UGACCCUUC 15 HumanCUGGAAGGGU AAGAUGUUGA [2361-2379] — CAACAUCUU CCCUUCCAG 16 HumanCCAGAGUGCU UGUAAUCCCA [2321-2339] — GGGAUUACA GCACUCUGG 17 HumanGAUGGGGUCU AACACUGUGA [2249-2267] — CACAGUGUU GACCCCAUC 18 HumanGCCAACUUUU AAAACAUGCA [2225-2243] — GCAUGUUUU AAAGUUGGC 19 HumanCCAUGGCCAG AAAAGUUGGC [2216-2234] — CCAACUUUU UGGCCAUGG 20 HumanAGACCCAGGU UUCAUCUGGA [288-306] — CCAGAUGAA CCUGGGUCU 21 Human,CCAUCAUCAC UCUUCCAGUG [878-896] [906-924] mouse ACUGGAAGA UGAUGAUGG 22Human, CAUCACACUG GGAGUCUUCC [882-900] [910-928] mouse GAAGACUCCAGUGUGAUG 23 Human, CAUCAUCACA GUCUUCCAGU [879-897] [907-925] mouseCUGGAAGAC GUGAUGAUG 24 Human, ACCAUCAUCA CUUCCAGUGU [877-895] [905-923]mouse CACUGGAAG GAUGAUGGU 25 Human, AUCAUCACAC AGUCUUCCAG [880-898][908-926] mouse UGGAAGACU UGUGAUGAU 26 Human, CACUGGAAGA CCACUGGAGU[887-905] [915-933] mouse CUCCAGUGG CUUCCAGUG 27 Human, ACACUGGAAGCACUGGAGUC [886-904] [766-784] [914-932] cynomoglus, ACUCCAGUG UUCCAGUGUmouse 28 Human, UCACACUGGA CUGGAGUCUU [884-902] [764-782] [912-930]cynomoglus, AGACUCCAG CCAGUGUGA mouse 29 Human, AUCACACUGG UGGAGUCUUC[883-901] [763-781] [911-929] cynomoglus, AAGACUCCA CAGUGUGAU mouse 30Human, CACAGCACAU ACCUCCGUCA [617-635] [497-515] [645-66] cynomoglus,GACGGAGGU UGUGCUGUG mouse 31 Human, CACUGGAAGA CCACUGGAGU [887-905][767-785] [915-933] cynomoglus, CUCCAGUGG CUUCCAGUG mouse 32 Human,UCACAGCACA CCUCCGUCAU [616-634] [496-514] [644-662] cynomoglus,UGACGGAGG GUGCUGUGA mouse 33 Human, GUCACAGCAC CUCCGUCAUG [615-633][495-513] [643-661] cynomoglus, AUGACGGAG UGCUGUGAC mouse 34 Human,CCAUCCACUA AUGUAGUUGU [812-830] [692-710] [702-720] cynomoglus,CAACUACAU AGUGGAUGG dog 35 Human, CCACCAUCCA UAGUUGUAGU [809-827][689-707] [699-717] cynomoglus, CUACAACUA GGAUGGUGG dog 36 Human,GAAUAUUUCA UCUGAAGGGU [1096-1114] [976-994]  [986-1004] cynomoglus,CCCUUCAGA GAAAUAUUC dog 37 Human, CGAGUGGAAG GCAAAUUUCC [706-724][586-604] [596-614] cynomoglus, GAAAUUUGC UUCCACUCG dog 38 Human,GAGAAUAUUU UGAAGGGUGA [1094-1112] [974-992]  [984-1002] cynomoglus,CACCCUUCA AAUAUUCUC dog 39 Human, CUACAUGUGU GGAACUGUUA [825-843][705-723] [715-733] cynomoglus, AACAGUUCC CACAUGUAG dog 40 Human,AACUACAUGU AACUGUUACA [823-841] [703-721] [713-731] cynomoglus,GUAACAGUU CAUGUAGUU dog 41 Human, CAACUACAUG ACUGUUACAC [822-840][702-720] [712-730] cynomoglus, UGUAACAGU AUGUAGUUG dog 42 Human,CACUACAACU UACACAUGUA [817-835] [697-715] [707-725] cynomoglus,ACAUGUGUA GUUGUAGUG dog 43 Human, CCACUACAAC ACACAUGUAG [816-834][696-714] [706-724] cynomoglus, UACAUGUGU UUGUAGUGG dog 44 Human,GACAGAAACA GUCGAAAAGU [742-760] [622-640] [632-650] cynomoglus,CUUUUCGAC GUUUCUGUC dog 45 Human, GGAGAAUAUU GAAGGGUGAA [1093-1111][973-991]  [983-1001] cynomoglus, UCACCCUUC AUAUUCUCC dog 46 Human,GUGUAACAGU CAUGCAGGAA [831-849] [711-729] [721-739] cynomoglus,UCCUGCAUG CUGUUACAC dog 47 Human, ACAACUACAU CUGUUACACA [821-839][701-719] [711-729] cynomoglus, GUGUAACAG UGUAGUUGU dog 48 Human,ACUACAACUA UUACACAUGU [818-836] [698-716] [708-726] cynomoglus,CAUGUGUAA AGUUGUAGU dog 49 Human, ACCAUCCACU UGUAGUUGUA [811-829][691-709] [701-719] cynomoglus, ACAACUACA GUGGAUGGU dog 50 Human,ACCACCAUCC AGUUGUAGUG [808-826] [688-706] [698-716] cynomoglus,ACUACAACU GAUGGUGGU dog 51 Human, UACCACCAUC GUUGUAGUGG [807-825][687-705] [697-715] cynomoglus, CACUACAAC AUGGUGGUA dog 52 Human,ACAGAAACAC UGUCGAAAAG [743-761] [623-641] [633-651] cynomoglus,UUUUCGACA UGUUUCUGU dog 53 Human, GAGUGGAAGG CGCAAAUUUC [707-725][587-605] [597-615] cynomoglus, AAAUUUGCG CUUCCACUC dog 54 Human,AUAUUUCACC GAUCUGAAGG [1098-1116] [978-996]  [988-1006] cynomoglus,CUUCAGAUC GUGAAAUAU dog 55 Human, AAUAUUUCAC AUCUGAAGGG [1097-1115][977-995]  [987-1005] cynomoglus, CCUUCAGAU UGAAAUAUU dog 56 Human,AGAAUAUUUC CUGAAGGGUG [1095-1113] [975-993]  [985-1003] cynomoglus,ACCCUUCAG AAAUAUUCU dog 57 Human, UGGAGAAUAU AAGGGUGAAA [1092-1110][972-990]  [982-1000] cynomoglus, UUCACCCUU UAUUCUCCA dog 58 Human,ACAUGUGUAA CAGGAACUGU [827-845] [707-725] [717-735] cynomoglus,CAGUUCCUG UACACAUGU dog 59 Human, UACAACUACA UGUUACACAU [820-838][700-718] [710-728] cynomoglus, UGUGUAACA GUAGUUGUA dog 60 Human,CUACAACUAC GUUACACAUG [819-837] [699-717] [709-727] cynomoglus,AUGUGUAAC UAGUUGUAG dog 61 Human, UCCACUACAA CACAUGUAGU [815-833][695-713] [705-723] cynomoglus, CUACAUGUG UGUAGUGGA dog 62 Human,AUCCACUACA ACAUGUAGUU [814-832] [694-712] [704-722] cynomoglus,ACUACAUGU GUAGUGGAU dog 63 Human, CAUCCACUAC CAUGUAGUUG [813-831][693-711] [703-721] cynomoglus, AACUACAUG UAGUGGAUG dog 64 Human,CACCAUCCAC GUAGUUGUAG [810-828] [690-708] [700-718] cynomoglus,UACAACUAC UGGAUGGUG dog 65 Human, UGUGUAACAG AUGCAGGAAC [830-848][710-728] [720-738] cynomoglus, UUCCUGCAU UGUUACACA dog 66 Human,CAUGUGUAAC GCAGGAACUG [828-846] [708-726] [718-736] cynomoglus,AGUUCCUGC UUACACAUG dog 67 Human, UACAUGUGUA AGGAACUGUU [826-844][706-724] [716-734] cynomoglus, ACAGUUCCU ACACAUGUA dog 68 Human,ACUACAUGUG GAACUGUUAC [824-842] [704-722] [714-732] cynomoglus,UAACAGUUC ACAUGUAGU dog 69 Human, AUCCGAGUGG AAUUUCCUUC [703-721][583-601] [593-611] cynomoglus, AAGGAAAUU CACUCGGAU dog 70 Human,UCACUCCAGC CUUCAGGUGG [1212-1230] [1092-1110] [1102-1120] cynomoglus,CACCUGAAG CUGGAGUGA dog 71 Human, CUCACUCCAG UUCAGGUGGC [1211-1229][1091-1109] [1101-1119] cynomoglus, CCACCUGAA UGGAGUGAG dog

Note that in the above Table B, the sense strands of siRNAs 1-71 haveSEQ ID NOS: 49-119 respectively, and the antisense strands of siRNAs1-71 have SEQ ID NOS: 120-190 respectively.

Table C below shows 63 additional 21-mer siRNAs which have beengenerated by the proprietary algorithms.

TABLE C gi2689466 gi5357emb gbU48957.1 X01237 gi4996229dbj gi13097806U48957 JMMP53R AB020761.1 Sense AntiSense gbBC003596.1 (Macaca (Mouse(Canis No. Source SiRNA SiRNA (Homo sapiens) fascicularis) mRNA)familiaris)  1 Human GGAAGAGAAUC UUCUUGCGGAG [975-995] — — — UCCGCAAGAAAUUCUCUUCC  2 Human GUACCACCAUC AGUUGUAGUGG [806-826] [686-706][835-852] [697-716] CACUACAACU AUGGUGGUAC  3 Human GGACGAUAUUGAACCAUUGUUC [261-281] — — — AACAAUGGUU AAUAUCGUCC  4 Human CCAGCCACCUGUUUUGGACUUC [1217-1237] [1097-1115] — [1107-1120] AAGUCCAAAA AGGUGGCUGG 5 Human GAGAAUAUUUC UCUGAAGGGUG [1094-1114] [974-994] [1122-1137] [984-1004] ACCCUUCAGA AAAUAUUCUC  6 Human AGAAACCACUG AUUCUCCAUCC[1079-1099] [959-979] — — GAUGGAGAAU AGUGGUUUCU  7 Human CUACUGGGACGAAAGCUGUUCC [910-930] [790-810] — — GAACAGCUUU GUCCCAGUAG  8 HumanAGACUCCAGUG AGUAGAUUACC [894-914] [774-794] [922-933] [784-795]GUAAUCUACU ACUGGAGUCU  9 Human CUGGAAGACUC AUUACCACUGG [889-909][769-789] [917-933] [779-795] CAGUGGUAAU AGUCUUCCAG 10 Human GAAACUACUUCUUGUUUUCAGG [189-209] [69-87] [235-247] [122-135] CUGAAAACAA AAGUAGUUUC11 Human GGAAACUACUU UGUUUUCAGGA [188-208] [68-87] [234-247] [122-134]CCUGAAAACA AGUAGUUUCC 12 Human AAACACUUUUC ACACUAUGUCG [747-767][627-647] — [637-657] GACAUAGUGU AAAAGUGUUU 13 Human GGAGUAUUUGGUUUCUGUCAUC [729-749] [609-629] — — AUGACAGAAA CAAAUACUCC 14 HumanUCAGACCUAUG AAGUAGUUUCC [178-198] [58-78] [231-244] — GAAACUACUUAUAGGUCUGA 15 Human CCAUGGCCAUC ACUGCUUGUAG [596-616] [476-496][624-640] [485-495] UACAAGCAGU AUGGCCAUGG 16 Human CCAAGUCUGUGACGUGCAAGUC [476-496] [356-376] — — ACUUGCACGU ACAGACUUGG 17 HumanGGACAGCCAAG AAGUCACAGAC [470-490] [352-370] [498-513] [357-377]UCUGUGACUU UUGGCUGUCC 18 Human CCCUCCUUCUC UAUAAAAAGGG [2421-2441] —[1721-1731] — CCUUUUUAUA AGAAGGAGGG 19 Human, CCAUCCACUAC ACAUGUAGUUG[812-832] [692-712] [840-860] [702-722] cynomoglus, AACUACAUGUUAGUGGAUGG dog 20 Human, CCACCAUCCAC UGUAGUUGUAG [809-829] [689-709][837-857] [699-719] cynomoglus, UACAACUACA UGGAUGGUGG dog 21 Human,GAGAAUAUUUC UCUGAAGGGUG [1094-1114] [794-994]  [984-1004] cynomoglus,ACCCUUCAGA AAAUAUUCUC dog 22 Human, GGAGAAUAUUU CUGAAGGGUGA [1093-1113][973-993]  [983-1003] cynomoglus, CACCCUUCAG AAUAUUCUCC dog 23 Human,CUACAUGUGUA CAGGAACUGUU [825-845] [705-725] [715-735] cynomoglus,ACAGUUCCUG ACACAUGUAG dog 24 Human, ACAACUACAUG AACUGUUACAC [821-841][701-721] [711-731] cynomoglus, UGUAACAGUU AUGUAGUUGU dog 25 Human,CCACUACAACU UUACACAUGUA [816-836] [696-716] [706-726] cynomoglus,ACAUGUGUAA GUUGUAGUGG dog 26 Human, CACCAUCCACU AUGUAGUUGUA [810-830][690-710] [700-720] cynomoglus, ACAACUACAU GUGGAUGGUG dog 27 Human,CAAUAUUUCAC GAUCUGAAGGG [1096-1116] [976-996]  [986-1006] cynomoglus,CCUUCAGAUC UGAAAUAUUC dog 28 Human, AGAAUAUUUCA AUCUGAAGGGU [1095-1115][975-995]  [985-1005] cynomoglus, CCCUUCAGAU GAAAUAUUCU dog 29 Human,UACCACCAUCC UAGUUGUAGUG [807-827] [687-707] [697-717] cynomoglus,ACUACAACUA GAUGGUGGUA dog 30 Human, GAUGGAGAAUA AAGGGUGAAAU [1090-1110][970-990]  [980-1000] cynomoglus, UUUCACCCUU AUUCUCCAUC dog 31 Human,AAGAGUGGAAG CGCAAAUUUCC [705-725] [585-605] [595-615] cynomoglus,GAAAUUUGCG UUCCACUCGG dog 32 Human, AACUACAUGUG GGAACUGUUAC [823-843][703-723] [713-733] cynomoglus, UAACAGUUCC ACAUGUAGUU dog 33 Human,CAACUACAUGU GAACUGUUACA [822-842] [702-722] [712-732] cynomoglus,GUAACAGUUC CAUGUAGUUG dog 34 Human, ACUACAACUAC UGUUACACAUG [818-838][698-718] [708-728] cynomoglus, AUGUGUAACA UAGUUGUAGU dog 35 Human,CACUACAACUA GUUACACAUGU [817-837] [697-717] [707-727] cynomoglus,CAUGUGUAAC AGUUGUAGUG dog 36 Human, UCCACUACAAC UACACAUGUAG [815-835][695-715] [705-725] cynomoglus, UACAUGUGUA UUGUAGUGGA dog 37 Human,CAUCCACUACA CACAUGUAGUU [813-833] [693-713] [703-723] cynomoglus,ACUACAUGUG GUAGUGGAUG dog 38 Human, ACCAUCCACUA CAUGUAGUUGU [811-831][691-711] [701-721] cynomoglus, CAACUACAUG AGUGGAUGGU dog 39 Human,UGGAGAAUAUU UGAAGGGUGAA [1092-1112] [972-992]  [982-1002] cynomoglus,UCACCCUUCA AUAUUCUCCA dog 40 Human, AUGUGUAACAG CAUGCAGGAAC [829-849][709-729] [719-739] cynomoglus, UUCCUGCAUG UGUUACACAU dog 41 Human,CAUGUGUAACA AUGCAGGAACU [828-848] [708-728] [718-738] cynomoglus,GUUCCUGCAU GUUACACAUG dog 42 Human, UACAACUACAU ACUGUUACACA [820-840][700-720] [710-730] cynomoglus, GUGUAACAGU UGUAGUUGUA dog 43 Human,CUACAACUACA CUGUUACACAU [819-839] [699-719] [709-729] cynomoglus,UGUGUAACAG GUAGUUGUAG dog 44 Human, AUCCACUACAA ACACAUGUAGU [814-834][694-714] [704-724] cynomoglus, CUACAUGUGU UGUAGUGGAU dog 45 Human,ACCACCAUCCA GUAGUUGUAGU [808-828] [688-708] [698-718] cynomoglus,CUACAACUAC GGAUGGUGGU dog 46 Human, AAUAUUUCACC GGAUCUGAAGG [1097-1117][977-997]  [987-1007] cynomoglus, CUUCAGAUCC GUGAAAUAUU dog 47 Human,ACUACAUGUGU AGGAACUGUUA [824-844] [704-724] [714-734] cynomoglus,AACAGUUCCU CACAUGUAGU dog 48 Human, AUGGAGAAUAU GAAGGGUGAAA [1091-1111][971-991]  [981-1001] cynomoglus, UUCACCCUUC UAUUCUCCAU dog 49 Human,UGUGUAACAGU CCAUGCAGGAA [830-850] [710-730] [720-740] cynomoglus,UCCUGCAUGG CUGUUACACA dog 50 Human, UCCGAGUGGAA GCAAAUUUCCU [704-724][584-604] [594-614] cynomoglus, GGAAAUUUGC UCCACUCGGA dog 51 Human,AUCCGAGUGGA CAAAUUUCCUU [703-723] [583-603] [593-613] cynomoglus,AGGAAAUUUG CCACUCGGAU dog 52 Human, UCACACUGGAA CACUGGAGUCU [884-904][764-784] [912-932] cynomoglus, GACUCCAGUG UCCAGUGUGA mouse 53 Human,AUCACACUGGA ACUGGAGUCUU [883-903] [763-783] [911-931] cynomoglus,AGACUCCAGU CCAGUGUGAU mouse 54 Human, CACACUGGAAG CCACUGGAGUC [885-905][765-785] [913-933] cynomoglus, ACUCCAGUGG UUCCAGUGUG mouse 55 Human,UCAUCACACUG UGGAGUCUUCC [881-901] [909-929] mouse GAAGACUCCA AGUGUGAUGA56 Human, CCAUCAUCACA AGUCUUCCAGU [878-898] [906-926] mouse CUGGAAGACUGUGAUGAUGG 57 Human, CAUCACACUGG CUGGAGUCUUC [882-902] [910-930] mouseAAGACUCCAG CAGUGUGAUG 58 Human, CAUCAUCACAC GAGUCUUCCAG [879-899][907-927] mouse UGGAAGACUC UGUGAUGAUG 59 Human, ACCAUCAUCAC GUCUUCCAGUG[877-897] [905-925] mouse ACUGGAAGAC UGAUGAUGGU 60 Human, UCACACUGGAACACUGGAGUCU [884-904] [912-932] mouse GACUCCAGUG UCCAGUGUGA 61 Human,AUCACACUGGA ACUGGAGUCUU [883-903] [911-931] mouse AGACUCCAGU CCAGUGUGAU62 Human, AUCAUCACACU GGAGUCUUCCA [880-900] [908-928] mouse GGAAGACUCCGUGUGAUGAU 63 Human, CACACUGGAAG CCACUGGAGUC [885-905] [913-933] mouseACUCCAGUGG UUCCAGUGUG

Note that in the above Table C, the sense strands of siRNAs 1-63 haveSEQ ID NOS: 191-253 respectively, and the antisense strands of siRNAs1-63 have SEQ ID NOS: 254-316 respectively.

Example 2 Testing the siRNA Compounds for Anti-p53 Activity Protocols I.Preparation of the siRNAs (Double-Stranded Oligonucleotides)

Lyophilized oligonucleotides were dissolved in RNAse free distilledwater to produce a final concentration of 100 uM. The dilutedoligonucleotides were kept at room temperature for 15 min andimmediately frozen in liquid nitrogen.

The oligonucleotides were stored at −80° C. and diluted before use withPBS.

II. Transfection of siRNA in Human Cells with Linofectamine2000 Reagent

2×10⁵ p53-wt HCT116 or SW480 cells were seeded per well in 6 wellsplate. 24 h subsequently, cells were transfected with p53oligonucleotides using lipofectamine2000 reagent (obtained fromInvitrogen).

The following procedure was performed:

-   -   1. Before transfection, the cell medium was replaced by 1500 ul        fresh medium without antibiotics.    -   2. In a sterile, plastic tube, Lipofecamine2000 reagent (the        amount is calculated according to 5 ul per well) was added to        250 ul serum-free medium, and incubated for 5 min at room        temperature.    -   3. In another tube the human anti-p53 oligonucleotides (varying        amounts to fit the desired final concentration per well) were        added to 250 ul serum-free medium.    -   4. Lipofectamine2000 complex was combined with the p53        oligonucleotide solution and incubated for 20 min at room        temperature.    -   5. The resulting mixture was added dropwise to the cells, and        the cells were incubated at 37° C.    -   6. SW480 cells: 48 hr after transfection the cells were        harvested and proteins were extracted using RIPA buffer.    -   7. HCT116 cells:        -   40 h after transfection, 5 Fu (Sigma) was added to cells to            produce a final concentration of 25 ug/ml. 48 h after cells            transfection (8 h after 5 Fu treatment), the cells were            harvested and proteins were extracted using RIPA buffer.    -   8. p53 expression was determined by Western Blot analysis using        monoclonal antibody (Do-1 clone, Santa Cruz). For normalization,        blots were examined for Tubulin expression.

III Co-Transfection of Mouse p53 Gene and Mouse p53 Oligonucleotidesinto PC3 Cells Using Lipofectamine2000 Reagent

2×10³ p53-null PC3 cells were seeded per well in 6 wells plate. 24 hsubsequently, cells were Co-transfected with mouse p53 gene and GFP geneand mouse p53 oligonucleotides using lipofectamine2000 reagent(Invitrogen). The following procedure was performed:

-   -   1. Before transfection cell medium was replaced by 1500 ul fresh        medium without antibiotics.    -   2. In sterile, plastic tube, Lipofectamine2000 reagent (5 ul per        well) was added to 250 ul serum-free medium, and incubated for 5        min at room temperature.    -   3. In another tube 4 ug DNA (p53 gene:GFPgene, 10:1) and human        p53 oligonucleotides were added to 250 ul serum free medium.    -   4. Lipofectamine2000 complex was combined with p53        oligonucleotides solution and incubated for 20 min at room        temperature.    -   5. The mixture solution was added dropwise to the cells, and        cells were incubated at 37° C.    -   6. 48 h after transfection, cells were harvested and proteins        were extracted using RIPA buffer.    -   7. p53 expression was determined by Western Blot analysis using        monoclonal antibody (Clone240, Chemicon). For normalization,        blots were examined for GFP expression.

Results:

A. Human p53 Oligonucleotides:

TABLE D Results of Test Number oligo species source SW480 HCT116 2 Hu2′human literature (−) (+) 3 QHMon1 human, Proprietary (++) (+++) monkey 4QHMon2 human, Proprietary (−) Not tested monkey 5 QH1 human Proprietary(+++) (+++) 6 QH2 human Proprietary (−) Not tested 13 A17 human, mouseProprietary (−) Not tested 14 E2 human, mouse, Proprietary (+) Nottested rat 15 E6 human, mouse, Proprietary (−) Not tested rat 16 B1human, mouse, Proprietary (−) Not tested rat 17 B2 human, mouse,Proprietary (−) Not tested rat 18 C1 human, Proprietary (−) Not testedmonkey, mouse 19 F2 human, Proprietary (−) Not tested monkey, dog 20 F3human, Proprietary (+++) (+++) monkey, dog 21 G1 human, Proprietary(+++) Not tested monkey, dog 22 H2 human, Proprietary (+) Not testedmonkey, dog 23 I5 human, Proprietary (+++) Not tested monkey, dog

Note: The numbers in Table D correspond to the numbers used in Table A,where the sense strands of siRNAs 1-23 have SEQ ID NOS: 3-25respectively, and the antisense strands of siRNAs 1-23 have SEQ ID NOS:26-48 respectively.

As shown in Table D, four human oligonucleotides were tested in twosystems SW480 and HCT116, according to Protocols II above.Representative results (Western Blot) on which the Results of Test wasbased are shown in FIG. 3.

B. Mouse p53 ollgouucleotides:

TABLE E Results of Test PC3 null cells/exogenous mouse oligo speciessource p53 1 Mo3 mouse literature (+++) 7 QM1 mouse Proprietary (−) 8QM2 mouse Proprietary (−) 9 QM3 mouse Proprietary (−) 10 QM6 mouseProprietary (−) 11 QM4 mouse, rat Proprietary (+++) 12 QM5 mouse, ratProprietary (+++) 13 A17 human, mouse Proprietary (−) 14 E2 human,mouse, rat Proprietary (++) 15 E6 human, mouse, rat Proprietary (−) 16B1 human, monkey, mouse Proprietary (−) 17 B2 human, monkey, mouseProprietary (++) 18 C1 human, monkey, mouse Proprietary (++) 19 G1human, monkey, dog Proprietary (++) 20 F3 human, monkey, dog Proprietary(+++) 21 I5 human, monkey, dog Proprietary (−) 22 QHMon1 human, monkeyProprietary (++)

Note: The numbers in Table E (as for Table D) correspond to the numbersused in Table A, where the sense strands of siRNAs 1-23 have SEQ ID NOS:3-25 respectively, and the antisense strands of siRNAs 1-23 have SEQ IDNOS: 26-48 respectively. Representatives of the Western Blot results onwhich the Results of Test was based are shown in FIG. 4.

Example 3 Model Systems of Hair Loss

Testing the active siRNA may be done in the following systems:

-   -   a. Mouse model of hair loss    -   b. Ex-vivo cultured human hair follicles    -   c. Human hair follicle graft in nude mice

Note: Systems for testing the active siRNA are described in Botcharev atal, 2000, p53 is essential for Chemotherapy-induced Hair Loss, CancerResearch 40, 5002-5006).

Example 4 Model Systems of Acute Renal Failure (ARF)

Testing the active siRNA for treating ARF may be done usingsepsis-induced ARF or ischemia-reperfusion-induced ARF.

1. Sepsis Induced ARF

Two predictive animal models of sepsis-induced ARF are described byMiyaji T, Hu X, Yuen P S, Muramatsu Y, Iyer S, Hewitt S M, Star R A,2003, Ethyl pyruvate decreases sepsis-induced acute renal failure andmultiple organ damage in aged mice, Kidney Int. November; 64(5):1620-31.These two models are lipopolysaccharide administration and cecalligation puncture in mice, preferably in aged mice.

2. Ischemia-Reperfusionsion-Induced ARF

This predictive animal model is described by Kelly K J, Plotkin Z.Vulgamott S L, Dagher P C, 2003 January, P53 mediates the apoptoticresponse to GTP depletion after renal ischemia-reperfusion: protectiverole of a p⁵³ inhibitor, J Am Soc Nephrol.; 14(1):128-38.

Ischemia-reperfusion injury was induced in rats following 45 minutesbilateral kidney arterial clamp and subsequent release of the clamp toallow 24 hours of reperfusion. 250 μg of p53 siRNA (QM5 sequence, TableA) were injected into the jugular vein 2 hrs prior to and 30 minutesfollowing the clamp. Additional 250 μg of siRNA were given via the tailvein at 4 and 8 hrs after the clamp. siRNA against GFP served as anegative control. The siRNA used in the experiments described herein hada phosphate group at the 3′ terminus of both the sense and antisensestrand. The 3′-non-phosphorylated siRNA has been found to have similarbiologically activity in an animal model as the corresponding3-phosphorylated siRNA. ARF progression was monitored by measurement ofserum creatinine levels before and 24 hrs post surgery. At the end ofthe experiment, the rats were perfused via an indwelling femoral linewith warm PBS followed by 4% paraformaldehyde. The left kidneys wereremoved and stored in 4% paraformaldehyde for subsequent histologicalanalysis. Acute renal failure is frequently defined as an acute increaseof the serum creatinine level from baseline. An increase of at least 0.5mg per dL or 44.2 μmol per L of serum creatinine is considered as anindication for acute renal failure. Serum creatiine was measured at timezero before the surgery and at 24 hours post ARF surgery.

To study the distribution of p53 siRNA in the rat kidney, Cy3-labeled19-mer blunt-ended siRNA molecules (2 mg/kg) having alternating O-methylmodification in the sugar residues were administered iv for 3-5 min,after which in vivo imaging was conducted using two-photon confocalmicroscopy. The confocal microscopy analysis revealed that the majorityof siRNA in the kidneys is concentrated in the endosomal compartment ofproximal tubular cells. Both endosomal and cytoplasmic siRNAfluorescence were relatively stable during the first 2 hrs post deliveryand disappeared at 24 bra.

As evident from FIG. 5, there was a ten-fold increase in the level ofserum creatinine following the 45-min of kidney bilateral arterial clamptreatment (PBS treatment). Four injections of p53 siRNA (QM5 sequence,Table A) (2 hrs prior to the clamp and 30 min, 4 h and 8 h after theclamp) significantly reduced the creatinine level in serum by 50%(P<0.001). These results suggest that p53 siRNA can protect renal tissuefrom the effects of ischemia-reperfusion injury and thus reduces theseverity of ARF.

The effect of p53 siRNA treatment on renal ischemia-reperfusion injurywas further determined by analysing the extent of tubular necrosis inthe renal tissue. Tubular necrosis may be scored as: no damage (damagescoring 0), unicellular, patchy isolated necrosis (damage scoring 1),tubular necrosis in less than 25% of the tissue (damage scoring 2),tubular necrosis in between 25 and 50% of the tissue (damage scoring 3)and tubular necrosis in more than 50% of the tissue (damage scoring 4).FIG. 6 demonstrates the tubular kidney damage expressed as damagescoring (Y-axis) in animals that did not undergo ischemia-reperfusioninjury (group 1) or in ischemia-reperfusion injury animals followingtreatment with either PBS (group 2), two injections of p53 siRNA (group3), three injections of p53 siRNA (group 4) or four injections of p53siRNA (group 5). As revealed by FIG. 6, four injections of p53 siRNA ledto significant decrease in the tubular kidney damage as compared to thePBS control group. FIG. 7 demonstrates that four injections of p53 siRNAtreatment down-regulated the expression of the pro-apoptotic gene Pumain the cortical compartment of the kidney in animal subjected toischemia-reperfusion injury. This indicates that p53 siRNA treatment iscapable of inhibiting the apoptotic processes in the kidney followingischemia-reperfusion injury.

1-43. (canceled)
 44. A method of treating a patient at risk ofdeveloping, or suffering from, acute renal failure comprisingadministering to the patient a double-stranded siRNA compound directedto a p53 gene in an amount effective to down-regulate the expression ofthe p53 gene so as to thereby treat the patient.
 45. The method of claim44, wherein the acute renal failure is ischemia-reperfusion-inducedacute renal failure.
 46. The method of claim 44, wherein the acute renalfailure is sepsis-associated acute renal failure.
 47. The method ofclaim 44, wherein the acute renal failure is toxic acute renal failuredue to chemotherapy treatment.
 48. The method of claim 44, wherein thepatient is a kidney transplant patient.